Method and kit for measuring micro-rna in body fluids

ABSTRACT

The present invention provides a kit for measuring the content of micro-RNA in a human blood sample including a blood collection tube containing at least 1 μg NaF and 0.8 μg KOx; and providing a set of instructions for collecting a human blood sample in the blood collection tube.

FEDERALLY SPONSORED RESEARCH

This work is supported by U.S. Army Medical Research and MaterielCommand under W81XWH-08-1-0641, NIH grant No. R01 CA119903 and NIH grantNo. T32-AI007392/AI/NIAID NIH HHS.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention provides a method and kit for measuring thequantity of microRNAs in human body fluids more accurately than priorart methodologies.

2. Background of the Invention

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers ofvarious diseases and conditions including malignancy and pregnancy. Thisapproach requires sensitive and accurate quantitation of miRNAconcentrations in body fluids. Enzyme-based miRNA quantitation, which iscurrently the mainstream approach for identifying differences in miRNAabundance among samples, is skewed by endogenous serum factors thatco-purify with miRNAs, and anticoagulants used during collection. Whatis important, different miRNAs were affected to different extents indifferent patient samples. The present invention provides a method andkit that overcome these interfering activities to increase the accuracyand sensitivity of miRNA detection in human body fluids up to 30-fold.

MicroRNAs are small, non-coding RNA sequences of about 19-22 nucleotidesthat function in modulating the activity of specific mRNA targets indevelopment, differentiation, or disease, typically by compromising mRNAstability or interfering with translation (reviewed in¹). Recently,miRNAs circulating in body fluids, and blood serum in particular, haveemerged as promising markers of disease and other processes (reviewedin¹⁻⁵). Accordingly, it is important to accurately identify andquantitate miRNAs in samples collected from patients.

The mainstream approach to identify and quantify miRNAs, utilizesquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).Using qRT-PCR, changes in plasma and serum miRNA profiles have beenreported to reflect various physiological and pathological conditions,including diagnostic and prognostic value for colorectal cancer, breastcancer, gastric cancer, leukemia, lung cancer, lymphoma, oral cancer,ovarian cancer, pancreatic cancer, prostate cancer (reviewed in¹⁻⁵) andother diseases or conditions⁶⁻⁹. The widening use of cell-freecirculating miRNA for diagnostic and prognostic purposes, as for anysuch marker, requires assurance that the measured concentrationrepresents the actual amounts in the samples. Such assurances are oftenlacking¹⁰. The problem is exacerbated by the common assumption that aprotocol developed for one study is applicable for others¹¹. Overall,few methods¹² and improvements¹³ have been offered¹⁴, and commonly usedapproaches have been shown to lack required accuracy¹⁵. CirculatingmicroRNAs (miRNAs) have emerged as candidate biomarkers of variousdiseases and conditions including malignancy and pregnancy. Thisapproach requires sensitive and accurate quantitation of miRNAconcentrations in body fluids. Here we report that enzyme-based miRNAquantitation, which is currently the mainstream approach for identifyingdifferences in miRNA abundance among samples, is skewed by endogenousserum factors that co-purify with miRNAs, and anticoagulants used duringcollection. Importantly, different miRNAs were affected to differentextents in different patient samples. By developing measures to overcomethese interfering activities, we increase the accuracy, and improve thesensitivity of miRNA detection up to 30-fold. Overall, our studyoutlines key factors that prevent accurate miRNA quantitation in bodyfluids and provides approaches that allow faithful quantitation of miRNAabundance in body fluids.

INTRODUCTION

MicroRNAs (miRNAs) are small non-coding RNA sequences of about 19-22nucleotides that function in modulating the activity of specific mRNAtargets in development, differentiation, or disease, typically bycompromising mRNA stability or interfering with translation (reviewedin¹). Recently, miRNAs circulating in body fluids, and blood serum inparticular, have emerged as promising markers of disease and otherprocesses (reviewed in¹⁻⁵). This application raises the need toaccurately identify and quantitate miRNAs in samples collected frompatients.

The mainstream approach to identify and quantify miRNAs, utilizesquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).Using qRT-PCR, changes in plasma and serum miRNA profiles have beenreported to reflect various physiological and pathological conditions,including diagnostic and prognostic value for colorectal cancer, breastcancer, gastric cancer, leukemia, lung cancer, lymphoma, oral cancer,ovarian cancer, pancreatic cancer, prostate cancer (reviewed in¹⁻⁵) andother diseases or conditions⁶⁻⁹. The widening use of cell-freecirculating miRNA for diagnostic and prognostic purposes, as for anysuch marker, requires assurance that the measured concentrationrepresents the actual amounts in the samples. Such assurances are oftenlacking¹⁰. The problem is exacerbated by the common assumption that aprotocol developed for one study is applicable for others¹¹. Overall,few methods¹² and improvements¹³ have been offered¹⁴, and commonly usedapproaches have been shown to lack required accuracy¹⁵.

In this study our goal was to standardize and optimize miRNA detectionfor biomarker studies. We quantified two miRNAs that are implicated indistinct processes. One was miR-16, which acts as a tumor suppressor, isUV-inducible, p53-regulated and is deregulated or lost in some cancers(reviewed in¹⁶). MiR-16 has also been used to normalize quantitation ofcirculating miRNAs for breast cancer studies¹⁷⁻¹⁹. The second miRNA,miR-223, has been implicated in pregnancy, other conditions andmalignancy^(6,20,21). Devising reliable approaches for accuratequantitation of circulating miRNAs is important in order to assess theirpotential as biomarkers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A,B Fluoride and Oxalate Increase the Quantitation Efficiency ofCirculating MicroRNAs. FIG. 1A MiR-16 detection is affected by bloodcollection method. Plasma and sera were collected from 4 individuals on3 separate occasions. MiR-16 was amplified using RT-PCR and separated byPAGE. Arrow: miR-16 as determined by sizing and sequencing. FIG. 1BCollection of blood in the presence of EDTA, citrate or fluoride andoxalate increases microRNA detection. MiR-16 and miR-223 were quantifiedusing SYBR green qRT-PCR approaches. Data were analyzed using one-wayanalysis of variance (ANOVA) followed by Tukey's HSD test. The middlebar represents the median, and the top and bottom of the box are the75th and 25th percentile. The Tukey fences indicate the range of thevalues. P values are labeled above or below brackets. Chemicals listedindicate the coagulant and/or blood stabilization agents present in theparticular VACUTAINER® used for the blood collection (Materials andMethods). Heparinase indicates the addition of heparinase I to thesample (Materials and Methods). Lower panel. MiR-223 detection is robustin NaF/KOx. Detection of miR-16 or miR-223 was less in heparinizedplasma than serum. Heparinase treatment did not always improve detectionof miR-16 or miR-223 in heparinized plasma (p=0.082 or p=0.125respectively). 50 bp and 60 bp indicate the DNA length of the bands inthe marker.

FIGS. 2A-C. Fluoride and Oxalate Improve Detection of RNA Regardless ofOrigin. FIG. 2) NaF/KOx was added at indicated concentrations into 50 μlof serum and plasma collected into EDTA. MiR-16 detection at bothconcentrations was increased (p=0.014 for both concentrations in serum,and p=0.037 for both concentrations in EDTA plasma. There was nostatistical difference between the two concentrations of NaF/KOx,p=0.827 for serum, and p=0.275 for EDTA plasma), (n=3). FIG. 2BSynthetic RNA (SYNTH was added at 250 fmoles per μl during the additionof Trizol to the serum samples or plasma collected using indicatedadditives. The effectiveness of SYNTH quantitation was compared to thatof endogenous miR-16. FIG. 2C) The amplification efficiency of miR-16and SYNTH in qPCR was measured and compared to that of serum (miR-16:Heparin: p=0.58, Heparin+Heparinase: p=0.43, EDTA: p=0.41, Citrate:p=0.52, KOx: p=0.43, SYNTH: Heparin: p=0.53, Heparin+Heparinase: p=0.49,EDTA: p=0.63, Citrate: p=0.41, KOx: p=0.47, n=3).

FIGS. 3A-E. Removal of Blood Plasma and Serum Components That InhibitDetection of Circulating MiRNA Using Organic Extraction and Silica-BasedRNA Enrichment. FIG. 3A Particulate miR-16 released from cells and addedto plasma or whole blood is not detectable within 17 hours. ParticulateRNAs released from BC3 cells was incubated in PBS, plasma or blood forindicated time, and miR-16 was quantified by TaqMan qRT-PCR. Portions ofmiR-16 in 2 h blood are likely of whole-blood origin. FIG. 3BPhenol/chloroform extraction and silica absorption of RNA removeinhibitors of miRNA detection. RNA was isolated from plasma collected inEDTA using Trizol only (left panel), or Trizol followed by oneextraction of phenol/chloroform and enrichment of small RNAs on a silicamembrane (right panel), and cDNA was added to a standard PCR reaction toquantify SYNTH RNA. RNA was added at the same concentration used in astandard qRT-PCR reaction (indicated by a “1” above the gel), and10-fold dilutions thereof (10⁻¹, 10⁻², 10⁻³). FIG. 3C Enhanced detectionof miR-16 in plasma extracted with phenol/chloroform and silicaabsorption. Plasma preparations were extracted using indicated numbersof phenol chloroform extractions, followed by absorption to silica, ordirectly assessed by qRT-PCR. FIGS. 3D, E Low volumes of plasma or serumeffect greater detection of miRNAs. Indicated volumes of serum extractedby Trizol were subjected to end-point PCR for miR-16 and separated byPAGE. Filled circles indicate amplification-independent products. MiR-16was quantified in EDTA plasma immediately after collection, or afterstorage at −80° C. SYBR Green or TaqMan.

FIGS. 4A-C. Heparin Interference with Detection of MiRNA is Relieved byHeparinase Treatment. Plasma was collected in VACUTAINER® blood tubescontaining sodium heparin, and indicated volumes of plasma were assessedby end-point PCR. FIG. 4A. RT-PCR was performed after treatment withHeparinase I (+) or on RNA without heparinase treatment (−). PCRproducts for miR-16 FIG. 4B and miR-223 FIG. 4C were assessed by PAGE.

FIGS. 5A-D. Mutant Taq DNA Polymerase, Hemo KlenTaq Improves theSensitivity of MicroRNA Detection. FIG. 5A Hemo KlenTaq (HK) was used todetect miR-16 (arrows) in 200 μl, 50 μl and 10 μl of plasma and serumused in FIG. 3C. Additional PCR products are marked with a star. FIG. 5BQuantitative TaqMan PCR of dilutions of reverse-transcribed miR-16 inserum or NaF/KOx plasma, and using an intact Taq polymerase (i) plusHemo KlenTaq (HKi) as indicated. FIG. 5C PAGE of miR-16 amplified withHKi or i from 6 individuals (1-6) and FIG. 5D absolute quantitation ofmiR-16 amplification products. ** indicates P<0.01, *** indicatesP<0.001 by Tukey-Kramer Multiple Comparisons test.

FIGS. 6A,B Screening of Taq and non-Taq DNA Polymerases in Serum-DerivedSamples. FIG. 6A The functionality of GoTaq, Phire, Phusion and HemoKlenTaq were tested using cDNA of miRNA isolated from the indicatedamount of starting volume of serum by endpoint PCR (40 cycles). (−)indicates PCR reaction was performed with Phire (left lane) or GoTaq(right lane) but with no template. FIG. 6B Quantitation by TaqMan.Detection of miR-16 was successful only for reactions using GoTaq.

FIG. 7. Components in Blood-Derived cDNA Produce Spurious PCRAmplification Products. Hemo KlenTaq was used to amplify miR-16 cDNAproduced from miRNAs released from BeWo cells into tissue culture media(BeWo exosomes). The cDNA was supplemented with dilutions ofplasma-derived cDNA (Blood cDNA (+)), or without additional cDNA.Copy-DNA products were used unpurified (1), or subjected to purificationusing ethanol precipitation (2), or a PCR clean kit (3).

FIGS. 8A,B. Titration of Hemo Klentaq With an Intact Taq Polymerase inTaqMan and SYBR Green Reactions Improves PCR Yield. FIG. 8A PAGEanalyses of end-point PCR products of reactions supplemented withindicated volume of Hemo KlenTaq in TaqMan or SYBR Green mastermixes asindicated. FIG. 8B, Quantitative PCR analyses of these reactions.Addition of 0.2 μl of Hemo KlenTaq to a 20 μl PCR reaction containingintact Taq polymerase was sufficient to amplify specific PCR productssuitable for qPCR by SYBR Green or TaqMan with increased amplificationefficiency.

FIGS. 9A,B Hemo KlenTaq Improves Detection of miR-16 in Plasma and SerumSamples. FIG. 9A Serial dilutions of oligos synthesized to reflectmiR-16 cDNA product during standard RT was quantified and plotted usingGoTaq alone or GoTaq and Hemo KlenTaq. GoTaq/Hem KlenTaq amplificationefficiency: 1.07+/−0.06 template duplications/cycle. Go Taq: 0.92+/−0.01template duplications/cycle). FIG. 9B Average cycle number at threshold.Samples 1-6 are the same as in FIG. 5D.

DETAILED DESCRIPTION OF THE INVENTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings, and will be described herein indetail, specific embodiments thereof with the understanding that thepresent disclosure is to be considered as an exemplification of theprinciples of the invention and is not intended to limit the inventionto the specific embodiments illustrated.

The present invention provides a method and a kit to standardize andoptimize miRNA detection for biomarker studies. We quantified two miRNAsthat are implicated in distinct processes. One was miR-16, which acts asa tumor suppressor, is UV-inducible, p53-regulated and is deregulated orlost in some cancers (reviewed in¹⁶). MiR-16 has also been used tonormalize quantitation of circulating miRNAs for breast cancerstudies¹⁷⁻¹⁹. The second miRNA, miR-223, has been implicated inpregnancy, other conditions and malignancy^(6,20,21).

In one preferred form of the invention, a kit is provided for measuringthe content of micro-RNA in a human blood sample. The kit includes ablood collection tube for collecting, for example, 5 ml of human blood.Blood collection tubes have colored tops that are standardized by allmanufacturers. Green tops designate the tube contains sodium or lithiumheparin, purple or lavender topped tubes contain EDTA, gray-topped tubescontain sodium fluoride and potassium oxalate and light-blue toppedtubes container sodium citrate. Commercially available VACUTAINER® bloodcollection tubes having gray closures contain 0.5 μg/μl and 0.4 μg/μlKOx.

In one preferred kit of the present invention, the quantity of NaF willbe from about 1.0 μg/μl to about 4.0 μg/μl and the quantity of KOx willbe from about 0.8 μg/μl to about 3.2 μg/μl in the blood collection tube.The NaF and KOx can be present in the tube prior to taking a bloodsample or added to the tube after the blood sample has been taken. Thekit will include a blood collection tube, a quantity of NaF and KOx inthe ranges set forth above and a set of instructions for using the kit.The kit will be packaged in a container appropriate for its purpose.

In another preferred form of the invention, a kit is provided for usewith a gray-topped blood collection container. The kit can optionallyinclude a gray-topped blood collection tube. The gray-topped tube isoptional as the kit can be used with a blood sample that has alreadybeen taken in a standard gray-topped blood collection tube. The kit willalso include a quantity of NaF and KOx to be added to the gray-toppedtube to increase the quantity of NaF and KOx to be within the desiredranges set forth above and preferably from about 0.5 μg/μl to about 3.5μg/μl and the quantity of KOx will be from about 0.4 μg/μl to about 2.8μg/μl. The NaF and KOx can be in separate containers, combined togetherin the same container or can be added to the gray-topped container priorto packaging. The NaF and KOx can be in powder form or in separatetablets or in a single tablet. The kit will also include a set ofdirections for using the kit with existing samples or with blood samplesto be taken and will be packaged in suitable packaging.

In another preferred form of the invention, a kit is provided for usewith heparin containing blood samples such as the green-topped bloodcollection containers. This kit will include a quantity of NaF and KOxin the desired amounts set forth above and in the desired powdered ortableted form. The kit will also include a quantity of heparinase I inpowder or tableted form to digest the heparin present in the bloodsample. The kit will include an appropriate set of instructions forusing the kit and will be packaged in suitable medical packaging.

The blood sample can be whole blood, serum or plasma.

The present invention further provides a method for collecting bloodfrom a human subject for measuring the quantity of miRNA in the sample.The method includes the steps of: (1) providing a sample of human bloodin a blood collection tube; (2) adding a quantity of NaF and KOx to theblood collection tube so that the blood collection tube has at least 1μg NaF and 0.8 μg KOx; (3) extracting miRNA from the blood collectiontube; and (4) quantifying the miRNA in the sample. In a preferred formof the invention, the human blood sample will have a volume of 5 ml andcan be whole blood, serum or plasma.

A preferred method for quantifying the miRNA is through a polymerasechain reaction procedure and particularly one using a combination ofHemo KlenTaq and an intact Taq polymerase. In one preferred form of theinvention, the Hemo KlenTaq was present in an amount by volume of fromabout 2% to about 8% of the volume of the PCR sample.

Materials and Methods

Fresh blood samples (5 ml) were collected from healthy adults, orreceived from the Susan G Komen Foundation for the Cure Tissue Bank atthe IU Simon Cancer Center (SGK samples), or supplied by Dr. JeffreyMartin from the AIDS Cancer and Specimen Resource (AIDS samples), SanFrancisco, Calif. Blood was collected in BD VACUTAINER® tubes containingheparin (sodium heparin, 143 usp unit, 10 ml), EDTA (EDTA 7.2 mg, 4.0ml), sodium citrate (sodium citrate, 0.105 M, 4.5 ml), or sodiumfluoride and potassium oxalate (sodium fluoride/potassium oxalate, 5mg/4 mg, 2 ml). Serum (7.5 ml) was collected in BD SSTT™, BDVACUTAINER®. Plasma was separated from red blood cells promptly toprevent loss of components²² or hemolysis²³. Blood was allowed tocoagulate for 15 min at room temperature prior to prompt centrifugation.All donors provided written consent and the work was approved by theRFUMS IRB under protocols #004 and #005 PATH.

Processing of Blood Samples

Fresh plasma and serum were obtained by centrifugation of blood samplesat 200 g for 15 min at 4° C. Supernatants were removed and collected in15 ml polypropylene tubes. The plasma was centrifuged twice at 800 g for15 min at 4° C. to obtain cell-free plasma. After the secondcentrifugation, supernatants were collected and passed through 0.45 μmpore-size filters (PALL, Port Washington, N.Y.). Plasma and serumsamples were divided into 200 μl, 50 μl and 10 μl samples, and totalvolumes were adjusted to 200 μl using water. A synthetic RNA, (SYNTH,formerly INT¹⁸, 250 fmol/μl) was added and samples were analyzedimmediately or flash-frozen.

Heparinase Treatment of Samples

Digestion of plasma samples with Heparinase I (Sigma-Aldrich, St. Louis,Mo.; H2519) was performed according to the manufacturer's protocol asfollows. Heparinase I (55 units) was dissolved at 1 mg/ml in 20 mMTris-HCl, pH 7.5, 50 mM NaCl, 4 mM CaCl₂, and 0.01% BSA and added to afinal concentration of 143 usp units per 10 ml of sample, which isexpected to nearly completely remove heparin²⁴. All digestions wereperformed for 1 h at room temperature, and heparinase was removed usingphenol-chloroform extractions.

Post-Collection Treatment of Samples with Sodium Fluoride and PotassiumOxalate

Potassium oxalate (Aqua Solutions, Deer Park Tex.; P5311, KOx) andsodium fluoride (Sigma-Aldrich, S-6776, NaF) were used for theseexperiments. Five ml blood was collected in NaF/KOx VACUTAINER® bloodTubes® containing 5 mg of NaF and 4 mg of KOx, effecting a finalconcentration of 1 μg/μl of NaF and 0.8 μg/μl KOx (1×). Therefore, 50 μlof serum and EDTA samples were supplemented with NaF/KOx at 8× (8 μg/μlNaF/6.4 μg/μl KOx), 4× (4 μg/μl NaF/3.2 μg/μl KOx), 2× (2 μg NaF/1.6μg/μl KOx), 1× (1 μg/μl NaF/0.8 μg/μl KOx), and 0.5× (0.5 μg/μl NaF/0.4μg/μl KOx) as indicated.

Determination of Exosomal miRNA Stability in Blood Plasma and WholeBlood

Blood was collected in EDTA BD VACUTAINER® blood Tubes®, and half wasprocessed to produce cell-free plasma, the other half was left intact.To test the stability in blood of miRNAs released from cells in culture,aliquots of the plasma and serum were supplemented with exosomal miRNAof BC3 cells, and incubated for the indicated amount of time at 10° C.in a continuously revolving tube rotator.

RNA Isolation

SDS was added to 200 μl samples of plasma/serum for a finalconcentration of 0.5% where indicated. This preparation was extractedwith 500 μl or 1 ml of Trizol LS reagent (Invitrogen) and incubated for10 minutes at room temperature followed by 100 or 200 μl of chloroform.The mixture was centrifuged at 12,000 g for 16 min and the aqueous layerwas transferred into a new tube. Where indicated, this preparation wasextracted with acidic phenol/chloroform (Amresco, Solon, Ohio; 0966) oneto three times. The resulting aqueous phase was transferred into a newtube and applied toPureLink™ miRNA isolation kit (Invitrogen, Carlsbad,Calif.) as indicated and processed according to the manufacturer'srecommendations. RNA was eluted with 50 μl of RNase-free water andstored at −70° C. or used immediately.

Cell Culture

Exosomes and other particulates were collected from cells in culture asdescribed¹⁸. In brief, after 5 days of culturing, the media wascollected, centrifuged at 300 g for 15 min, and filtered through a 0.45μm filter to remove cell debris. The supernatant was centrifuged at70,000 g to collect particulates including exosomes, and resuspendedwith 100 μl PBS. BeWo cells were purchased from ATCC (CCL-98, Manassas,Va., USA) and cultured in F-12K or RPMI 1640 (Mediatech Inc. Manassas,Va. or Hyclone Logan, Utah respectively) with 10% or 20% fetal bovineserum (FBS) respectively. To remove bovine particulates, includingexosomes, FBS was ultracentrifuged at 70,000 g for 2 h, and thecollected supernatant was added to culture media. BC3 cells werecultured as described²⁵.

Reverse Transcription (RT)

For studies with fresh plasma, serum and SGK samples, 10 μl of the 50 μlextracted RNA was used as input into a Superscript III (Invitrogen,Carlsbad, Calif.) reverse transcriptase reaction with miRNA-specificstem-loop primers in the Duelli laboratory as described¹⁸ ²⁶. Thethermal cycles used to amplify the samples was 65° C. for 5 min, 50° C.for 60 seconds, 70° C. for 15 seconds for 40 cycles. AIDS samples andsamples including miRNAs released from BC3 cells were analyzed usingprimers from Applied Biosystems, Inc., ABI (Foster City, Calif.), alsousing miRNA-specific stem-loop primers in the Cullen laboratory asdescribed²⁶ ²⁷.

MicroRNA Quantitation by Taq-Based PCR

Quantitative PCR reactions were performed as described using SYBR® Greenor TaqMan® (ABI) as noted¹⁸. Four percent of the cDNA produced in the RTreaction was amplified in MicroAmp™ Optical 96-well reaction plates intriplicate 20 μl reactions on an Applied Biosystems 7900HT Thermocycler(ABI) using the cycle 95° C., 10 min; 40 cycles of 95° C., 15 sec and60° C. for 1 min; and hold at 4° C. Raw data was analyzed with SDSRelative Quantitation Software version 2.2.3 (ABI), generally using theautomatic cycle threshold (Ct) setting for assigning baseline andthreshold for Ct determination. MiRNA abundance was measured bycomputing amoles based on comparing CT values of samples to dilutions ofa synthetic DNA corresponding to the cDNA produced by RT for each miRNAmeasured to make a standard curve. The amplification efficiency, ameasure of number of template duplications per PCR amplification cyclewas calculated using the equation (T2/T1)^((1/(CT2 ave-CT1 ave)))−1²⁸.

PCR with Other Polymerases

GoTaq® Green (Promega, Madison, Wis.) PCR was performed in 25 μl volumesaccording to manufacturer's instructions. The initial denaturation stepwas 5 min at 95° C., followed by 40 cycles of 15 seconds at 95° C., 30seconds at 50° C., 30 seconds at 72° C. and a final extension of 5 minat 73° C.

Hemo KlenTaq™²⁹ (New England BioLabs, Ipswich, Mass.) PCR¹⁸ wasperformed in 25 μl volumes according to manufacturer instructions,including attempts to reduce non-specific priming by assembling PCRreactions on ice and transfer of reactions to the thermocycler preheatedto 95° C. PCR using cocktails containing GoTaq® DNA polymerase and HemoKlenTaq™ polymerase were performed using GoTaq® qPCR Mastermix (Promega)for SYBR Green quantitation or TaqMan® Universal PCR Mastermix (ABI).

Phire and Phusion enzymes were used according to supplier specifications(New England Biolabs) for end-point PCR, or used with GoTaq® qPCRMastermix (Promega) for SYBR Green or with TaqMan Universal PCRMastermix (ABI) for quantitation.

Page

Native polyacrylamide gel electrophoresis (PAGE) of PCR products wasperformed as described¹⁸. A 10 base-pair (bp) ladder (Invitrogen,Carlsbad, Calif.) was used for sizing PCR products in all experiments(Marker (M)). Typically, 8 μl of each PCR sample was analyzed by PAGE.

Statistical Analysis

Statistical analysis was performed by one-way analysis of variance(ANOVA) followed by Tukey's Honestly Significant Difference (HSD) testwhere indicated. Column graphed data are presented as +/− one standarddeviation of the mean. Data points were compared by the unpairedone-tailed t test unless otherwise indicated, and the P-values arelabeled in the figures, text or in the legend, as are the number ofindependent experiments.

Results

NaF and KOx improve MicroRNA Quantitation

Different anticoagulants and blood stabilizers are used for plasmacollection, but serum is collected without stabilizers oranticoagulants. Therefore, we tested whether the choice of collectionmethod affected miRNA quantitation. We collected blood into bloodevacuation tubes containing one of the following anticoagulants: EDTA,heparin, sodium citrate (Citrate), sodium fluoride and potassium oxalate(NaF/KOx), or collected blood in the absence of anticoagulants. Bloodwas collected into the different blood evacuation tubes in immediatesuccession using a single venipuncture per person (Materials andMethods), to ensure near-identical blood composition at the time ofblood collection. Blood was drawn from four individuals of differentethnic origin, sex and age on three separate dates.

We measured miRNA abundance by reverse-transcription PCR (RT-PCR) (FIG.1A), and quantitative PCR (qPCR) of reverse-transcribed miRNAs with SybrGreen (FIG. 1B). We found that reproducibility of miRNA quantitationdepended on the blood collection methods, with best results obtained bycollecting into the tubes containing NaF/KOx (FIG. 1B). Although miR-16is about 500-fold more abundant than miR-223 in blood plasma, therecovery and accurate detection of both miRNAs depended on the plasmacollection method. Therefore, these differences in detection andquantitation resulting from the choice of blood collection methods arelikely to occur in measuring other circulating miRNAs, regardless ofabundance. Furthermore, quantitation of miR-223 in serum yielded morevariable results than most other collection methods, suggesting that atleast for some miRNAs, collection of blood as plasma, and the choice ofanticoagulants can improve detection.

NaF and KOx Improve the Sensitivity of MicroRNA Detection Post-BloodCollection

Maintenance of RNA stability during and after blood collection is animportant factor for accurate miRNA quantitation and may depend on theanticoagulant used to collect the sample. For example, NaF/KOx isthought to allow greater sensitivity in the quantitation of othermolecules in the blood, including glucose²², alcohol and opioids³⁰, bypreventing their degradation at the time of collection and duringstorage³¹. We reasoned that the anticoagulants may differentially affectmiRNA stability, or detection by interfering with, or promotingreactions used for quantitation. Therefore, we evaluated the effect thatNaF and KOx have on the quantitation of miRNAs collected by other blooddraw methods. We added NaF and KOx to frozen samples collected by two ofthe most common blood draw methods, serum and plasma collected intoEDTA, and measured miR-16 in these samples (Materials and Methods). Wefound that NaF and KOx increased miR-16 detection 2-fold in plasmacollected using EDTA (p=0.037) and 3-fold in serum (p=0.014)respectively (FIG. 2A). These results indicate that adding NaF and KOxenhances miRNA quantitation even if these reagents are added after thesamples were collected using other anticoagulants and stabilizers.

To address how NaF and KOx improve miRNA quantitation, we assessed theeffect that NaF and KOx have individually on miR-16 quantitation inserum. NaF increased the mean detection of miR-16 2.8 fold, and KOxincreased sensitivity about 3.4 fold. However, the increases lackedstatistical significance for each component alone, suggesting that NaFand KOx synergize to effectively increase detection of miRNAs, or thatthey act differently in the improvement of quantitation of miRNAs inserum.

NaF and KOx Increase the Detection of Exogenous RNA

We considered that collecting plasma with NaF and KOx can improvedetection by 1) stabilizing the RNA, or 2) increasing the amplificationefficiency of the miRNAs during the PCR reaction^(29,32). To address thepossibility that miRNAs are degraded by plasma ribonucleases³³, wequantified miRNA stability prior to miRNA extraction by comparingendogenous miR-16 concentrations to that of a synthetic 22 nucleotideRNA (SYNTH) added during the RNA extraction. We found that the quantityof RNA was similar for both SYNTH RNA and the endogenous miR-16 for eachof the different blood collection containers (FIG. 2B), suggesting thatfactors other than RNA stability in serum or plasma account for thedifferences in the sensitivity of RNA quantitation. We therefore testedwhether the collection method affects the PCR amplification efficiency.We detected no significant changes in the amplification efficienciesinduced by the blood stabilizers or anti-coagulants (FIG. 2C). Weconclude from this result that none of the blood collection methodsaffect the PCR amplification of reverse-transcribed miRNAs per se. HenceNaF and KOx may improve miRNA detection by enhancing miRNA yield duringthe extraction, by enhancing the reverse transcriptase reaction, orperhaps in general, by stabilizing the extracted RNA or the cDNA.

The finding of strong parallels between SYNTH and miR-16 quantitationwithin each plasma and serum sample support the convention of usingexogenous spiked RNAs as reference molecules for quantifying miRNA inplasma and serum. Alternatively, we found that quantitation of SYNTHadded in a 4000-fold excess was much less affected by most collectionmethods. This result suggests that high concentrations of RNA canovercome the interference of plasma components on miRNA quantitation,and highlights the need to supply spiked RNA at similar concentrationsas the endogenous RNA under investigation for accurate referencequantitation.

Inhibitors of Polymerases Present in Biological Samples Affect MicroRNADetection

To assess the stability of miRNAs in blood, we added released miRNAsisolated from the media of cultured BC3 cells to PBS, plasma and wholeblood. We found that miR-16 could be detected up to 17 h after additionto PBS, but was undetectable in plasma after 2 h or whole blood after 17hours of storage (FIG. 3A). This destabilization effect could be due tomiRNA degradation in the sample³⁴, or because of components of bloodplasma that co-purify with miRNAs and interfere with theirdetection^(29,35-37), or both.

Therefore, we tested whether plasma RNA interferes with the detection ofsynthetic SYNTH RNA. To do so, we supplemented SYNTH RNA withTrizol-extracted RNA preparations of plasma collected in EDTAVACUTAINER® blood tubes, and quantified SYNTH RNA abundance (FIG. 3B,left panel). We found that RNA extracted from blood plasma interferedwith SYNTH RNA detection, indicating that inhibitors of reversetranscriptase or PCR are present in RNA preparations extracted withTrizol alone.

To determine the best method for removing serum and plasma inhibitors ofmiRNA detection, we applied approaches to improve the purity of theisolated RNA. We found that the incorporation of a single acidicphenol/chloroform extraction step followed by adsorption of RNA onsilica membranes reduced the interference by blood-borne polymeraseinhibitors (FIG. 3B, right panel), and effected a 4.4-fold increase inmiRNA quantitation (FIG. 3C). Other treatments, such as the addition ofdetergents³⁸ or ribonuclease inhibitor RNAsin did not improve miRNAdetection in serum or plasma suggesting that components other thanribonucleases or other proteins were responsible for the interference.We conclude that enrichment of small RNAs using Trizol andphenol/chloroform extractions and silica-adsorption effectively removeblood-borne RT and/or PCR inhibitors that prevent accurate quantitationof miRNAs present in blood plasma.

Plasma Volume Affects MiRNA Detection and Quantitation

The presence of inhibitors of miRNA detection in blood suggests that thegreater the plasma or serum starting material used to extract the miRNA,the greater the abundance of co-purified blood-borne inhibitors ofRT-PCR. Thus, we tested whether dilution of starting material affectsthe efficiency of detection. To do so, we quantified miRNA extractedfrom 10 μl, 50 μl or 200 μl of fresh serum or plasma. We found that 50μl of serum improved detection of miRNA by end-point PCR (FIG. 3D), andyielded an 11-fold increase in the sensitivity of miRNA detection bySYBR Green or TaqMan qPCR (FIG. 3E), perhaps reflecting a balancebetween miRNA and inhibitor abundance. To test if similar concentrationeffects also apply to stored samples, we tested plasma collected intoEDTA from HIV and KSHV infected patients, and stored for several months.We found that, similar to fresh samples, detection of miR-16 abundancewas about 3-fold more sensitive at 50 μl than 10 μl or 200 μl (FIG. 3E).These data are consistent with the counter-intuitive idea that usingmore blood for detecting miRNA actually results in less efficientdetection than using less blood. Because this effect was seen in bothplasma and serum, the latter of which is collected in the absence ofadditives, we concluded that inhibitors of miRNA detection are inherentto blood, rather than introduced by chemicals used for collection.Importantly, by reducing the starting material, inhibitors werepresumably diluted below a threshold of interference. Thus, carefultitration of starting material yields more accurate miRNA quantitation.

Heparinase Treatment of Plasma Increases microRNA Detection.

Heparin is an endogenous component of blood, and one of the originalanti-coagulants used in medicine. In some instances, for example whenanalyzing historical samples, or evaluating the blood of patients onheparin regimens used for deep venous thromboses, strokes, pulmonaryembolism, during organ transplantation or heart surgery, heparinizedplasma may be the only source of miRNA. In these cases, it will beimportant to evaluate the effect of heparin on miRNA quantitation. Wefound that miRNA quantified from heparinized plasma gave a poor yield(FIG. 1), consistent with the fact that RT-PCR is inhibited byheparin³⁹. We tested whether reducing the starting volume could improvedetection. We found that using less starting material allowed detectionof miR-16 from heparinized blood. However, greater dilutions wererequired to effect similar detection as in plasma collected by othermethods (compare FIG. 3D to FIG. 4A). We therefore tested whether miRNAdetection can be improved by digesting heparin with lyase heparinase Iprior to RT-PCR^(24,40). Strikingly, this treatment allowed thedetection and quantitation of miRNAs that were previously undetectable(FIGS. 4B and 4C). We conclude that heparinase can increase detection ofmiRNAs in heparinized plasma. Importantly, these results suggest thatheparin tubes should be avoided when possible for miRNA analyses.Alternatively, heparinase treatment provides an approach to detectmiRNAs in cases where the use of heparin was unavoidable or when heparinis present in previously collected samples.

A Mutant Taq DNA Polymerase Improves Quantitation of MicroRNA.

An alternative approach to avoid interference from blood-borneinhibitors of RT-PCR is to use different polymerases^(29,35-37,41). Wetested enzymes reported to be more resistant to inhibitors: Phusion,Phire, and Hemo KlenTaq. Hemo KlenTaq is a mutant Taq DNA polymerase,which reportedly has a 100-fold lower sensitivity to blood inhibitorsthan wild-type Taq²⁹. Our analysis indicates that Hemo KlenTaq amplifiedmiR-16, more efficiently than Phusion (FIGS. 5A and 6) and standard TaqDNA polymerase yielded low or no detectable PCR products in the samesamples (FIG. 3D). However, initial attempts to adopt these enzymes forqPCR of miRNAs using TaqMan failed (FIG. 6B). Likely, for Hemo KlenTaqthe quantitation was compromised by the production of multiple spuriousbands in addition to the correct band (FIGS. 5A and 6A) We tested ifthis lack of specificity is a general property of Hemo KlenTaq, byevaluating the purity of amplifying miR-16 released from BeWo cells inculture. We detected only the correct, miR-16 PCR product. However,supplementing such PCR reactions with blood plasma cDNA was sufficientto give rise to the extra products, regardless of attempts to purifycDNA further prior to PCR amplification (FIG. 7). This finding suggeststhat the complexity or other properties of blood cDNA interfere with theexclusive amplification of the miRNAs of interest and cause non-specificcDNA amplification. This phenomenon may be a consequence of the lack ofthe editing- and proofreading 5′->3′ exonuclease domain in HemoKlenTaq³².

A Complementing Enzyme Cocktail for Effective Amplification of Products

To overcome the limitations of Hemo KlenTaq, we tested whether thereduced proof-reading activity of Hemo KlenTaq could be complemented byan intact Taq polymerase. We found that Hemo KlenTaq in combination withintact Taq polymerases amplified specific PCR products suitable for qPCRby SYBR Green or TaqMan (FIGS. 5B, 5C and 8A). Importantly, thecomplementing Taq polymerases produced about 30 times more amplificationproducts from plasma miR-16 on average than the intact polymerase alone(FIG. 8B) suggesting that overcoming blood-borne inhibitors of Taqpolymerase using Hemo KlenTaq yield an overall increase in detectionsensitivity and specificity of circulating miRNAs.

Mir-16 Quantitation Sensitive to Method of Detection

To test if overcoming endogenous inhibitors with complementingpolymerases provides proportionally higher miRNA quantitation, weanalyzed circulating miRNAs in the blood of six healthy individuals(FIGS. 9A,B) Five of the six individuals tested show similar miR-16plasma concentrations, regardless of approach used, thus validating theuse of complementing enzymes for improved detection of circulatingmiRNAs. Interestingly, one individual in each approach consistently hadhigher plasma miR-16 abundance than the others (FIG. 5D). However, adifferent individual in either approach had higher miR-16concentrations, suggesting that the quantitation of circulating miR-16,which is used as a reference miRNA or a biomarker of some cancers¹⁶⁻¹⁹,depends on the PCR conditions. These results suggest that differences inplasma composition among individual donors can yield different miRNAmeasurements.

TABLE 1 Effectiveness in MiRNA Detection With Different TreatmentsTreatment/ Extra Sensitivity/ Approach Sensitivity¹ Time² Cost³ Cost⁴FIGS. Choice of anticoagulant and stabilizer NaF/KOx  2.7 0 99% 2.7 1EDTA  1.1 0 99% 1.1 1 Citrate  1.7 0 100% 1.7 1 Heparin  0.3 0 101% 0.31 Serum  1 0 100% 1.0 1 NaF/KOx post-  3.6⁵ seconds 100% 3.6 2Acollection Starting volume  10 μl  1 0 100% 1.0 3D, E, 4A  50 μl 11⁶ 0100% 11 3D, E, 4A 200 μl  0.7 0 100% 0.7 3D, E, 4A Silica/Phenol/  4.411 min⁷ 181% 2.4 3B, C Chloroform Hemo Klentaq/Go 30 seconds 101% 30 5Taq Heparinase  2⁸  1 h 130% 1.5 1, 4 treatment ¹Approximate folddifference in detected abundance compared to serum miR-16. ²Per sample.³Material cost per sample: Calculated as % of total cost of serum miR-16detection using TaqMan (in $, based on list prices of material usedwithin study, which was $9.25 for triplicate TaqMan) ⁴Increase inSensitivity returned per Material investment. ⁵2.4-fold in EDTA plasma.⁶3-fold in frozen samples. ⁷the extra total time spent on adsorption persample is gained by elimination of the chilling step for precipitationof RNA (1 h plus) in the standard method. However, there is an increasein active labor, which, depending on the number of samples processed,may by substantial. ⁸Fold of plasma collected in heparin. Ranges fromstatistically non-significant changes (FIG. 1) to detecting signal onlyafter heparinase treatment (FIG. 4).

Discussion

This study demonstrates that inherent differences in biological samplesand the methods used to collect and analyze them can dramatically affectthe detection and quantitation of miRNAs. The implications of the workare that without consideration of the variables we have identified,miRNA quantitation from human samples may not be reliable for thepurpose of biomarker development. Our results suggest that failure todetect plasma miRNAs may be due to polymerase inhibitors rather than theactual absence of miRNA. Such inhibitors may include hemoglobin⁴²,lactoferrin³⁶, and immunoglobulin G⁴³, which can co-purify with nucleicacids³⁷. This limitation can be overcome by the concomitant use of twocomplementing Taq polymerases; Hemo KlenTaq, which is resistant toblood-borne inhibitors, in combination with another intact polymerasethat has effective proof-reading ability. We also demonstrate thatdiluting out inhibitors from blood samples also provides salientimprovements in miRNA detection (Table 1). Additional purification ofplasma or serum miRNA preparations, using organic extraction and silicaadsorption to remove inhibitors, also increased the detection, albeit ata greater cost in labor and funds.

The inability to detect specific miRNAs in plasma or serum, in manycases, reflects the low abundance of particular miRNAs in thecirculation. For example, the release of some miRNAs from cells intoblood is limited or selective^(18,44,45). Furthermore, depending on thenature of the complex circulating miRNAs are associatedwith^(18,34,46-52), some miRNAs may be more stable than others³⁴ to thedegradation by plasma ribonucleases, or may be more amenable toamplification by polymerases than others. The improvements outlined hereallow the quantitation of miRNAs with very low abundance, where usualtechniques fail. The use of these approaches is expected to increase therepertoire of miRNAs that can be analyzed as potential biomarkers ofdisease.

The mechanism by which some of the approaches, for example the useNaF/KOx improve detection is unclear. Possibly, NaF effects higher cDNAstability during the reverse transcriptase reaction, rather thanstabilize input miRNA, because classical experiments identified NaF asan inhibitor of RNAse H⁵³, an enzyme that degrades RNA/DNA hybridsubstrates. On the other hand, KOx may promote miRNA detection byreducing calcium, a known inhibitor of Taq, in the blood sample⁴¹.

Interestingly, the absolute quantity of circulating miR-16 measured insome individuals' blood was different depending on the enzyme used.These differences raise the possibility that factors, including diet⁵⁴,exercise⁵⁵, circadian rhythms⁵⁶ and seasons^(57,58), which alter theblood chemistry might ultimately affect miRNA detection andquantitation. The disparate effectiveness of miRNA detection inindividual's plasma or sera may be indicative of other, physiologic andpathogenic properties of the blood, including endogenous heparinconcentrations⁵⁹ or familial disease⁶⁰. Our results demonstrate thatsuch variations in blood chemistry can affect the detection of miRNAs,and must be considered and neutralized in order to accurately andefficiently assess miRNA abundance in blood serum and plasma.

From the foregoing, it will be observed that numerous variations andmodifications may be effected without departing from the spirit andscope of the invention. It is to be understood that no limitation withrespect to the specific apparatus illustrated herein is intended orshould be inferred. It is, of course, intended to cover by the appendedclaims all such modifications as fall within the scope of the claims

What is claimed is:
 1. A kit for measuring the content of micro-RNA in ahuman blood sample comprising: a blood collection tube containing atleast 1 μg/μl NaF and 0.8 μg/μl KOx; and a set of instructions forcollecting a human blood sample in the blood collection tube.
 2. The kitof claim 1 wherein the blood collection tube is free from heparin.
 3. Akit for measuring the content of micro-RNA in a human blood samplecomprising: a blood collection tube; a quantity of NaF and KOx foradding to the blood collection tube so that the collection tube has atleast 1 μg/μl NaF and 0.8 μg/μl KOx; and a set of instructions forcollecting human blood sample in the collection tube.
 4. The kit ofclaim 3 further comprising: a quantity of heparinase I for adding to theblood collection tube.
 5. A method for collecting blood from a humansubject comprising the steps of: providing a sample of human blood in ablood collection tube; adding a quantity of NaF and KOx to the bloodcollection tube so that the blood collection tube has at least 1 μg/μlNaF and 0.8 μg/μl KOx; extracting miRNA from the blood collection tube;and quantifying the miRNA in the sample.
 6. The method of claim 5wherein the human blood has a volume of 5 ml.
 7. The method of claim 5wherein the blood sample is whole blood, serum or plasma.
 8. The methodof claim 7 wherein the step of quantifying the miRNA is through apolymerase chain reaction procedure.
 9. The method of claim 8 whereinthe polymerase chain reaction procedure utilizes Hemo KlenTaq and anintact Taq polymerase.
 10. The method of claim 5 further comprising thestep of adding heparinase Ito the collection tube.